...The Evo­lu­tion of Ster­il­iza­tion Tech­niques

by Ananya Sen

His­tory is rife with tech­niques and ma­te­ri­als used to pre­vent mi­cro­bial growth. The Egyp­tians are famed for their process of mum­mi­fi­ca­tion, which pre­serves the skin and or­gans of de­ceased bod­ies. They cir­cum­vented pu­tre­fac­tion by de­hy­drat­ing the body and us­ing chem­i­cals such as na­tron (a salt) that ef­fec­tively ster­il­izes the body and pre­vents bac­te­r­ial de­com­po­si­tion (sim­i­lar to how beef jerky is made). There are sev­eral other ex­am­ples: Greeks used sil­ver and cop­per con­tain­ers to store food and wi­ne, ex­plor­ers like Christo­pher Colum­bus traded spices be­cause of their use as preser­v­a­tives (or to dis­guise bad tastes), and sol­diers in the Amer­i­can Rev­o­lu­tion were gi­ven three pints of spruce beer every day be­cause of the lack of potable wa­ter. Fur­ther­more, hypochlor­ous acid and hy­dro­gen per­ox­ide were used as house­hold dis­in­fec­tants long be­fore it was known that the im­mune sys­tem uses those same chem­i­cals to de­stroy in­vad­ing patho­gens.

Fig­ure 1. War ar­mor- this was worn by doc­tors who treated plague. The beak was stuffed with herbs to keep away the bad odors that were be­lieved to the cause of in­fec­tious dis­eases. Source

The main pur­pose of dis­in­fec­tion and ster­il­iza­tion, then and now, is to counter the spread of mi­crobes, es­pe­cially pathogens. Al­though the causative mech­a­nism of dis­ease pro­gres­sion was un­known, sci­en­tists pos­tu­lated a con­nec­tion be­tween pu­tre­fac­tion and the dis­se­mination of dis­ease. There­fore, they set out to elim­i­nate the ef­fects of de­com­po­si­tion to re­duce the in­ci­dence of in­fec­tions. These at­tempts led to a mul­ti­pronged ap­proach. I will trace the his­tory of each ap­proach sep­a­rately.

Chem­i­cal com­bat

One of the first for­mal stud­ies on ster­il­iza­tion was car­ried out by Dutch mer­chant and sci­en­tist An­tonie van Leeuwen­hoek in 1676. Peer­ing into his mi­cro­scopes, Leeuwen­hoek re­al­ized that che­mi­cals such as pep­perand wine-vine­gar caused bac­te­r­ial death, as evinced by their loss of motil­ity. His work was fol­lowed up by Eng­lish physi­cian Ed­mund King (in 1693), who added sul­fu­ric acid, salt, sugar, wine, ink, and blood to the list of chem­i­cals that could be used as ster­il­iza­tion agents. The 18th cen­tury saw the dis­cov­ery of chlo­rine in 1774 by the Swedish chemist Carl Scheele and the dis­cov­ery of hypochlo­rites in 1789 by the French chemist Claude Berthol­let. These chem­i­cals had the dis­tinc­tion of coun­ter­ing the odors as­so­ci­ated with pu­tre­fac­tion. Un­for­tu­nately, they rein­forced the mis­taken be­lief that dis­ease was caused by de­cay and nox­ious smells.

Fig­ure 2. Lis­ter­ine was de­vel­oped in 1879 by Joseph Lawrence in honor of Joseph Lis­ter. It was orig­i­nally de­vel­oped as an al­co­hol-based sur­gi­cal an­ti­sep­tic. Source

It was Louis Pasteur's work from 1860 – 1864 that ce­men­ted the germ the­ory of dis­ease. Fol­low­ing that, mi­cro­bio­lo­gists scoured their sur­round­ings to find chem­i­cals that could be used to kill mi­crobes. In 1865 Joseph Lis­ter tes­ted phe­nol as a dis­in­fec­tant to treat a frac­ture wound. He chose phe­nol be­cause he had heard of its abil­ity to pre­vent the odor of sewage in a nearby vil­lage. How­ever, the credit of us­ing phe­nol is not Lister's alone. He had been un­aware of the works of sev­eral oth­ers (Kuchen­meis­ter in 1860, Lemaire in 1860) that had shown that phe­nol is ef­fec­tive in treat­ing wounds (And that is why it is im­port­ant to read cur­rent sci­en­tific lit­er­a­ture!). To Lister's credit, he suc­ceeded in con­vinc­ing sev­eral sur­geons to adopt his an­ti­sep­tic tech­nique; his per­sua­sive­ness can be partly at­tributed to his suc­cess in treat­ing Queen Victoria's armpit sore with phe­nol. He also worked out the con­cen­tra­tions of phe­nol that were suf­fi­cient to treat wounds, since un­di­luted phe­nol is highly caus­tic and causes in­flam­ma­tion of tis­sues. In 1881 Robert Koch pub­lished a de­tailed re­port on the prepa­ra­tion meth­ods for over 70 chem­i­cals that could be used to kill an­thrax spores, in­clud­ing io­dine, potas­sium per­man­ganate, formic acid, qui­nine, and oil of tur­pen­tine. The fi­nal cor­ner­stone in es­tab­lish­ing the prin­ci­ples of chem­i­cal dis­in­fec­tion was the work done by Kro­nig and Paul in 1897. They rec­og­nized that bac­te­ria are not all killed si­mul­ta­ne­ously; the rate of killing be­ing de­pen­dent on the con­cen­tra­tion of the chem­i­cal and the tem­per­a­ture used. These prin­ci­ples led to the de­vel­op­ment of the fa­mous phe­nol co­ef­fi­cient test for dis­in­fec­tants (Rideal and Walker, 1903). This test is a mea­sure of the bac­te­ri­ci­dal ac­tiv­ity of a chem­i­cal in re­la­tion to phe­nol us­ing an ar­bi­trary con­tact time (7.5 min­utes), a dilu­ent (dis­tilled wa­ter), and a stan­dard test or­gan­ism (they used Sal­mo­nella ty­phi). A mod­i­fied ver­sion of this pro­to­col is now the ba­sis for test­ing mod­ern dis­in­fec­tants.

Some like it hot

One of the ear­li­est records of us­ing heat ster­il­iza­tion comes from 1810, by Parisian chef Nico­las Ap­pert. In an at­tempt to pre­serve food, he put it in glass jars, which he sealed and placed in boil­ing wa­ter. This sim­ple method be­came wide­spread and right­fully earned him the ti­tle "the fa­ther of can­ning". In 1837 a Ger­man physi­cian, Theodor Schwann, used heat ster­il­iza­tion to dis­prove spon­ta­neous gen­er­a­tion: when ster­ile sugar so­lu­tions were ex­posed to air, the sug­ars de­com­posed, but no de­com­po­si­tion oc­curred when the air was heat-ster­il­ized. Years later, in 1860, Louis Pas­teur con­clu­sively proved that heat could de­stroy mi­croor­gan­isms us­ing his fa­mous swan-neck ex­per­i­ment. Sub­se­quently he in­sisted that sur­geons pass their in­stru­ments through a flame to de­stroy ad­her­ing mi­crobes, and ad­vo­cated the use of heat-ster­il­ized ban­dages on open wounds. Pas­teur is also cred­ited with de­vel­op­ing pas­teur­iza­tion, a method that pre­vents the spoil­ing of milk, beer, and wine by heat­ing them briefly at 50°C – 60°C.

Fig­ure 3. Papin's steam di­gester that served as a model for the mod­ern au­to­clave. Source

The pri­mary dis­ad­van­tage of pas­teur­iza­tion is that it does not kill all the mi­crobes; it only re­duces the num­ber of vi­able pathogens. Try­ing to achieve steril­ity, British phy­si­cist John Tyn­dall de­vel­oped the process of Tyn­dal­liza­tion in 1877. Here, the liq­uid to be ster­il­ized was heated at 100°C for 30 min­utes on three suc­ces­sive days. The first round killed the veg­e­ta­tive cells, the sec­ond round killed the veg­e­ta­tive cells that had ger­mi­nated from endo­spor­es, and the third round was a pre­cau­tion­ary mea­sure. This was a te­dious process, and the re­sults were not al­ways re­pro­ducible. This prob­lem was solved in 1879 with the de­vel­op­ment of the au­to­clave by Charles Cham­ber­land. He mod­eled the au­to­clave us­ing the prin­ci­ples of the high-pres­sure cooker in­vented by French physi­cist De­nis Pa­pin in 1679. Au­to­clav­ing sub­jects the ma­te­ri­als to high pres­sure steam at 121°C for 15 – 20 min­utes. This method is able to de­stroy all bac­te­ria (ex­cept some ex­treme ther­mophiles), viruses, fungi, and spores, and is still the pri­mary method of ster­il­iza­tion to­day.

Razedby Ra­di­a­tion

The first sci­en­tific study on the ef­fect of light on mi­crobes was car­ried out in 1877 by Downes and Blunt. They showed that ex­pos­ing so­lu­tions of sugar wa­ter to sun­light pre­vented mi­cro­bial growth. They fur­ther demon­strated that this phe­nom­e­non was de­pen­dent on the in­ten­sity, du­ra­tion, and wave­length of the light used; the shorter wave­lengths of the so­lar spec­trum were the most ef­fec­tive. This re­sult was con­firmed a year later by Tyn­dall, who no­ticed that when cul­tures were ex­pos­ed to sun­light there was no bac­te­r­ial growth. How­ever, when these cul­tures were sub­se­quently moved to warmer tem­per­a­tures, they did grow. This ob­ser­va­tion led him to hy­poth­e­size that light in­hib­ited bac­te­r­ial growth in­stead of caus­ing de­creased vi­a­bil­ity. How­ever, he did not con­sider that the high con­cen­tra­tion of bac­te­ria in his flask was prob­a­bly pro­tect­ing some cells from sun­light dam­age. In 1885 Duclaux and Ar­loing demon­strated the killing ef­fect of sun­light us­ing pure cul­tures of Bacil­lus an­thracis; the spores of these bac­te­ria were un­able to ger­mi­nate af­ter pro­long­ed ex­po­sure to sun­light. Even so, the re­gion of the spec­trum that was re­spon­si­ble for the killing ac­tion re­mained un­known.

In 1892 Theodor Geisler demon­strated that UV rays were the most ef­fec­tive in killing Sal­mo­nella ty­phi. He in­ves­ti­gated the ef­fect of the vis­i­ble spec­tra on bac­te­r­ial growth us­ing a prism and a he­lio­stat; he found that UV rays caused the most dam­age by us­ing ura­nium-glass tubes which al­lowed UV rays to pass through. Be­tween 1893–95 Mar­shall Ward un­equiv­o­cally proved that the vi­o­let-blue and near UV rays were the most harm­ful to bac­te­ria. He built on the ex­per­i­ments of Geisler and metic­u­lously con­structed lens sys­tems that al­lowed for max­i­mal sta­bil­ity of illu­mi­na­tion. He showed that there was growth in­hi­bi­tion in the UV-vi­o­let-blue re­gion with a sharp de­cline at the bor­der of blue and green light. This ef­fect was not ob­served with any other re­gion of the vis­i­ble or in­fra-red spec­trum. How­ever, none of these stud­ies quan­ti­fied the in­ten­sity of the light source re­quired to kill bac­te­ria. Such a study was car­ried out by Her­tel in 1904, who proved that the dele­te­ri­ous ef­fects of ra­di­a­tion were in­versely pro­por­tional to the wave­length of light and di­rectly pro­por­tional to the dose (in­ten­sity x du­ra­tion) of ra­di­a­tion. All these stud­ies con­tributed sig­nif­i­cantly to pub­lic health poli­cies re­gard­ing wa­ter ster­il­iza­tion and re­duced the in­ci­dence of in­fec­tious dis­eases.
 

Fil­tra­tion- the non-vi­o­lent line of de­fense

Fig­ure 4. Orig­i­nal map by John Snow show­ing the clus­ters of cholera cases in the Lon­don epi­demic of 1854. The pump is lo­cated at the in­ter­sec­tion of Broad Street and Cam­bridge Street. Source

Fil­tra­tion was prac­ticed as early as 4000 B.C.. An­cient Greek and San­skrit writ­ings rec­om­mended fil­ter­ing wa­ter through char­coal. These at­tempts were in­tended to re­duce the amount of vis­i­ble cloudi­ness (tur­bid­ity) in drink­ing wa­ter. The first for­mal study of the ben­e­fits of fil­tra­tion was car­ried out by Fran­cis Ba­con (posthu­mously pub­lished in 1627). who was try­ing to de­sali­nate sea wa­ter. His ex­per­i­ments laid the foun­da­tion for us­ing sand fil­ters to re­move par­tic­u­lates from wa­ter. In 1804 John Gibb used sand fil­ters in or­der to sup­ply fil­tered wa­ter to res­i­den­tial ar­eas in the U.K., thus cre­at­ing the first treated pub­lic wa­ter sup­ply in the world. There­after prac­tice of fil­tra­tion be­came com­mon­place. Its virtues were ce­mented by John Snow dur­ing the 1854 Broad Street cholera out­break. His sta­tis­ti­cal stud­ies showed that there was a link be­tween the qual­ity of wa­ter source and the in­ci­dence of cholera. This con­vinced the au­tho­ri­ties to dis­able the wa­ter pumps which ended the out­break. The ma­te­ri­als used to cre­ate fil­ters in­cluded cot­ton wool (Schröder and von Dusch 1854) and ce­ram­ics (Cham­ber­land 1884). The fi­nal demon­stra­tion that fil­tra­tion led to steril­ity was car­ried out by Tyn­dall in 1876 – 1877. He showed that if the dust and mi­cro-or­gan­isms were re­moved by fil­tra­tion, the air would not con­t­a­m­i­nate ster­ile so­lu­tions that were left ex­posed to it. The so­lu­tions be­came con­t­a­m­i­nated when left open to air that con­tained par­tic­u­late mat­ter. The fil­ters used to­day can re­move 99.97% of 0.3 mm par­ticles, which in­cludes most bac­te­r­ial cells but not viruses. Nanofil­ters with a pore size of 20 – 50 nm are used to re­move viruses.

Cur­rent meth­ods of ster­il­iza­tion

Cur­rent meth­ods of ster­il­iza­tion

The meth­ods of ster­il­iza­tion em­ployed nowa­days vary based on the place of use. In food and dairy in­dus­tries, dis­in­fec­tion and san­i­ti­za­tion are more im­por­tant than to­tal ster­il­iza­tion. Con­versely, in lab­o­ra­tory stud­ies equip­ment and me­dia need to be ster­ile to pre­vent any con­t­a­m­i­na­tion. Some of the var­i­ous meth­ods of ster­il­iza­tion have been sum­ma­rized be­low.

 

Ananya is a grad­u­ate stu­dent in the De­part­ment of Mi­cro­bi­ol­ogy at the Uni­ver­sity of Illi­nois at Ur­bana-Cham­paign. She works in the lab of James A. Im­lay. Ananya has re­cently started a blog of her own called " The His­tory of Sci­ence."